Study on the Application of Doxorubicin-Loaded Magnetic Nanodrugs in Targeted Therapy of Liver Cancer

Doxorubicin (DOX) has been widely recognized as effective in anticancer therapy; however, the additional organ toxicity and low targeting of DOX in antitumor activity remains unaddressed. The aim of this study was to use the drug carrier dextran/polylactic acid (DEX/PLA) and paramagnetic Fe3O4 to improve the targeting of DOX in liver cancer treatment while reducing its potential organ toxicity. Our experimental results show that the combination drug (Fe3O4@DEX/PLA-DOX) exhibits strong hepatocarcinogenic inhibition and low cytotoxicity and that this modified drug provides a better option for clinical liver cancer treatment.


Introduction
Liver cancer is one of the six common cancers in the world. It can be divided into primary liver cancer and secondary liver cancer. Primary liver cancer refers to the primary tumor of the liver, and secondary liver cancer refers to other parts of the liver. The tumor has metastasized to the tumor in the liver [1][2][3][4]. The incidence of liver cancer is extremely high, and it is also the fourth leading cause of cancer deaths. And the survey shows that the incidence of liver cancer is higher in underdeveloped areas. East Asia, South Asia, and North Asia are all high-risk areas for liver cancer [5]. The causes of liver cancer are still unclear. Alcoholism, obesity, cirrhosis, hepatitis B, hepatitis C, fatty liver, and diabetes are all risk factors for liver cancer [6,7]. In terms of treatment, liver resection and liver transplantation have always been the first choice for the treatment of liver cancer [8,9]. In addition, chemotherapy, radiotherapy, molecular targeted therapy, immunotherapy, antiviral therapy, traditional Chinese medicine, and other adjuvant sexual therapy can effectively inhibit liver cancer. Among them, radiotherapy is not a first-line treatment plan for liver cancer, but it can be used as an effective means for local treatment of liver cancer [10][11][12]. Chemotherapy is a common treatment method in traditional cancer treatment, but it is more harmful to the liver, and it is easy to aggravate the symptoms of liver cirrhosis and hepatitis. It is currently commonly used in the treatment of advanced liver cancer [13]. Antiviral therapy mainly targets liver cancer caused by hepatitis B and C. At the same time, it can also inhibit the replication of the hepatitis virus caused by Feilaozi-targeted drugs and chemotherapy drugs. It can be used throughout the treatment of liver cancer [14][15][16]. Immunotherapy can enhance the body's own immune function, break immune tolerance, and stimulate the body's tumor-specific immunity to delay tumor development [17]. Molecular targeted therapy has now occupied an important position in the treatment of liver cancer. Compared with other therapeutic drugs, it has low toxicity and high selectivity. It can target the disease through a variety of signaling pathways, which is the focus of current research [18][19][20]. In addition, traditional Chinese medicine for the treatment of liver cancer is gradually being recognized by the public, and studies have shown that it can effectively inhibit the recurrence and metastasis of liver cancer.
Doxorubicin (DOX), as an anthracycline antibiotic, can be trimerized between DNA base pairs, triggering topoisomerase II to cleave DNA, thereby causing damage to the tertiary structure of DNA [21]. In addition, as a cell cycle nonspecific drug, it can cause cytotoxic effects on the cells at various stages, so it is widely used in the treatment of breast cancer, lung cancer, liver cancer, ovarian cancer, bladder cancer, and other cancers. However, DOX usually causes high fever, nausea, vomiting, phlebitis, bone marrow transplantation, and severe cardiotoxicity [22]. Therefore, how to reduce the occurrence of side effects plays an important role in clinical applications. Targeted nanoformulation is a drug formulation that can identify specific targets of the lesion and can be targeted for delivery. It can usually be delivered by carriers such as liposomal microspheres, microcapsules, and nanoparticles. It can improve the problem of poor solubility of drugs and at the same time reduce the damage of drugs to other parts except for the lesion [23]. Magnetic nanoparticles can achieve targeted drug delivery for liver cancer by loading drugs on carriers containing magnetic materials. Common magnetic targeting materials include ferrite magnetic materials, Fe3O4 iron powder, and magnetic alloy materials [24].
Dextran (DEX) is a water-soluble polysaccharide with a linear backbone, which has good biocompatibility and nontoxic effects. In addition, DEX has good reactivity, can load a variety of biologically active molecules, and has good excipient characteristics when used in the modification of magnetic nanoparticles, which can effectively promote application research in tumors and immunodetection [25]. Polylactic acid (PLA) is a bio-based renewable and degradable material with a wide range of sources. It is widely used in packaging materials, fibers, clothing, construction, agriculture, and medical and health fields. It is a good drug carrier material [26]. Based on this, this study used DOX as a therapeutic drug and DEX/PLA as a drug carrier, modified with Fe3O4 to obtain Fe3O4@DEX/PLA-DOX and study its inhibitory effect on liver cancer.  2.3.2. Preparation of DEX/PLA Vector. 500 mg of PLA, 200 mg of N-N ′ -dicyclohexylcarbodiimide, and 15 mg of 4dimethylaminopyridine were placed in the flask, 30 mL of DMSO was added, and the mixture was stirred and dissolved at 60°C. Continue the reaction for 30 min. Immediately add 250 mg of DEX, and react for two days under nitrogen protection. The reaction mixture was put into a dialysis bag and dialyzed in running water for 48 h. The suspension was centrifuged at 12,000 rpm for 20 min, the supernatant was lyophilized and dissolved in acetone solution for washing, and the solid lyophilized was obtained by suction filtration to prepare the DEX/PLA carrier.

Preparation of Fe 3 O 4 @DEX/PLA-DOX.
Fe 3 SO 4 magnetic nanoparticles were dissolved in DMSO, added DEX/ PLA carrier, stirred uniformly at 60°C, and reacted for 24 h. When the reaction was over, put them in a dialysis bag and dialyze with double-distilled water for 48 h. Centrifuge the suspension in the dialysis bag at 3,000 rpm for 10 min. The supernatant was lyophilized and dissolved in an acetone solution. The solid after suction filtration was washed and then lyophilized to obtain Fe 3 O 4 @DEX/PLA graft things.
The preparation of Fe 3 O 4 @DEX/PLA-DOX adopts the solvent diffusion method. Dissolve DOX in double-distilled water and add it to Fe 3 O 4 @DEX/PLA graft solution in DMSO at a ratio of 1 : 20, and transfer it to dialysis. The inside of the bag was dialyzed with double-distilled water for 20 h in the dark and then centrifuged at 3,000 rpm for 10 min. The supernatant was collected to obtain the Fe 3 O 4 @DEX/PLA-DOX solution. Use electron microscopy to characterize and measure its particle size at the same time.  On the 21st day after the administration, 6 mice in each group were sacrificed. The tumor is removed and weighed. The remaining mice were fed naturally to death, and their average survival time was observed. Its therapeutic mechanism for liver cancer is shown in Figure 1.

Targeting
Effect. The blood, heart, liver, kidney, and tumor tissues of the sacrificed mice were frozen and ground, added with saline, mixed well, and centrifuged at 12,000 rpm for 20 min. The supernatant was used to detect the main tissues in the HPLC content of DOX.

Fe 3 O 4 @DOX-DEX/PLA Morphology Characteristics.
Fe 3 O 4 @DEX/PLA-DOX nanoparticles were characterized by electron microscopy, and the characterization results are shown in Figure 2. Its appearance is spherical, with an average particle size of 86:31 ± 10:68 nm. Because its particle size is less than 100 nm, it has high permeability and can freely enter and exit the tumor site with a multivascular structure, which is beneficial to the treatment of cancer.

Encapsulation Rate and Drug
Loading Rate. Figure 3 shows  3.6. Targeting. In order to investigate the targeting of Fe 3 O 4 @DEX/PLA-DOX to liver cancer, the DOX content in the main organs of mice in the Fe 3 O 4 @DEX/PLA-DOX group and the DOX group was detected (Figure 7). The results showed that the Fe 3 O 4 @DEX/PLA-DOX content in the tumor was up to 7:31 ± 1:02 μg/g, while the DOX group was only 2:53 ± 0:27 μg/g. This proves that Fe 3 O 4 @DEX/ PLA-DOX nanoparticles are targeted for liver cancer. In addition, since the DOX content in other main organs of mice in the Fe 3 O 4 @DEX/PLA-DOX group is lower, it can effectively avoid other organ damage caused by DOX.

Discussion
As an antibacterial and cytostatic drug, DOX is widely used in the treatment of malignant tumors [27]. Its targeted preparation can improve the inhibitory effect on tumors and reduce the toxic effect of DOX on nonfocal sites. It is a promising new way of administration [28]. With the increasing incidence of liver cancer, DOX targeting agents have also become a hot spot in current research [29,30]. The ideal targeted preparation for inhibiting liver cancer can concentrate all the drugs on the lesion, but the current    The characterization results showed that Fe 3 O 4 @DEX/ PLA-DOX nanoparticles were characterized, and the characterization results showed that Fe 3 O 4 @DEX/PLA-DOX has a spherical appearance and an average particle size of less than 100 nm. It has high permeability and can be used in the tumor site with a multivascular structure that can enter and exit freely, which is conducive to the treatment of cancer. Meanwhile, the encapsulation rate, drug loading rate, and in vitro drug release experiment results proved that it can effectively coat DOX and accelerate its release under acidic conditions to effectively inhibit cancer tissues. In vitro toxicity experiments proved that the carrier of Fe 3 O 4 @DEX/ PLA-DOX is not cytotoxic and has good biological safety. Compared with DOX, Fe 3 O 4 @DEX/PLA-DOX has a better

Conclusion
In this work, Fe3O4@DEX/PLA-DOX magnetic nanoparticles were prepared by using DEX/PLA as the drug carrier and modifying DOX with Fe3O4. The results of the in vivo and ex vivo experiments on structural characterization, encapsulation rate, drug loading rate, drug release capacity, toxicity, targeting, and ability to inhibit hepatocellular carcinoma found that Fe3O4@DEX/PLA-DOX has certain targeted inhibitory effect on hepatocellular carcinoma, and its inhibitory effect on hepatocellular carcinoma is stronger compared with that of DOX alone. The findings of this study are expected to provide a new way for the clinical treatment of liver cancer.

Data Availability
The data underlying the results presented in the study are available within the manuscript.

Ethical Approval
Research experiments conducted in this article with animals were approved by the Medical Ethics Committee of Qingdao Jiaozhou Central Hospital following all guidelines, regulations, and legal and ethical standards as required for animals.

Conflicts of Interest
There are no conflicts to declare.   Applied Bionics and Biomechanics